9-ING-41, a small molecule inhibitor of GSK-3beta, potentiates the effects of anticancer therapeutics in bladder cancer

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase, has been identified as a potential therapeutic target in human bladder cancer. In the present study, we investigated the antitumor effect of a small molecule GSK-3β inhibitor, 9-ING-41, currently in clinical studies in patients with advanced cancer, in bladder cancer cell lines. We found that treatment with 9-ING-41 leads to cell cycle arrest, autophagy and apoptosis in bladder cancer cells. The autophagy inhibitor chloroquine potentiated the antitumor effects of 9-ING-41 when tested in combination studies. Our findings also demonstrate that 9-ING-41 enhanced the growth inhibitory effects of gemcitabine or cisplatin when used in combination in bladder cancer cells. Finally, we found that 9-ING-41 sensitized bladder cancer cells to the cytotoxic effects of human immune effector cells. Our results provide a rationale for the inclusion of patients with advanced bladder cancer in clinical studies of 9-ING-41.

suppresses proliferation of bladder cancer cells (Fig. 1B). Our finding that 9-ING-41 inhibits proliferation of bladder cancer cells prompted us to examine the effect of 9-ING-41 on cell cycle kinetics. We found cell cycle blockage at G2/M after 24 hours of 9-ING-41 treatment (Fig. 1C), suggesting that GSK-3 inactivation by 9-ING-41 halts progression of mitosis in bladder cancer cells. To investigate the mechanistic effect of GSK-3 inhibitor 9-ING-41 in the blockage of cell cycle in bladder cancer cells, we examined the expression of G2/M regulatory proteins Cdk1 and Cyclin B1 in 9-ING-41-treated cells. We found that expression of Cdk1 and Cyclin B1 proteins was significantly decreased in 9-ING-41-treated bladder cancer cells ( Fig. 2A). Moreover, treatment with 9-ING-41 led to a decreased expression of antiapoptotic molecules, Bcl-2 and XIAP, resulted in an increased apoptosis as shown by PARP cleavage in bladder cancer cells ( Fig. 2A,B). Furthermore, we used caspase activation assay to demonstrate that 9-ING-41 treatment induces apoptotic cell death in bladder cancer cells (Fig. 2C). Our in vitro results suggest that treatment with GSK-3 inhibitor 9-ING-41 suppresses expression of G2/M regulatory proteins (B) T24 and HT1376 bladder cancer cells were treated with 9-ING-41 for 72 hours and mRNA expression of Bcl-2 and XIAP was analyzed by RT-PCR. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test. (C) Activity of caspase-3 was detected in 9-ING-41-treated bladder cancer cells using colorimetric CaspACE Assay System. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test.

Inhibition of autophagy potentiates antitumor effects of 9-ING-41 in bladder cancer cells.
Despite the extensive investigation on the role of autophagy in tumor growth and metabolism, it is still unclear whether autophagy supports or inhibits viability of cancer cells 19 . In cancer cells, autophagy could be induced by different antitumor therapies [20][21][22] . We noticed significant morphological changes in 9-ING-41-treated bladder cancer cells (Fig. 3A). After 24 hours treatment with 9-ING-41, T24 cancer cells showed extensive vacuolation and formation of autophagosome like structures in the cytoplasm (Fig. 3A). The development of autophagy was confirmed by detection of an increased expression of LC3, an autophagy marker, in 9-ING-41-treated bladder cancer cells (Fig. 3B). Moreover, we found that combination of 9-ING-41 and chloroquine, an autophagy inhibitor, significantly suppressed viability of T24 (P < 0.05) and HT1376 (P < 0.05) cancer cells as compared to the effects of 9-ING-41 or chloroquine monotherapy (Fig. 3C). These results suggest that inhibition of autophagy could potentiate the antitumor effect of 9-ING-41 in bladder cancer cells.

9-ING-41 potentiates antitumor effect of gemcitabine and cisplatin in bladder cancer cells. It
has been demonstrated that GSK-3 positively regulates NF-κB-mediated survival and chemoresistance of cancer cells 23 . Using chemoresistant p53-mut T24 and HT1376 bladder cancer cell lines, we tested our hypothesis that 9-ING-41 may overcome resistance to standard of care chemotherapeutic drugs in bladder cancer. Because the plasma half-life of 9-ING-41 is approximately 3 hours, we treated bladder cancer cells for 3 hours with 2 μM 9-ING-41 and chemotherapeutic drugs. Post-treatment, all test compounds were replaced with fresh cell culture medium. Then, bladder cancer cells were allowed to grow for 72 hours and relative cell growth was evaluated by MTS assay after 72 hours of cell incubation in drug-free culture medium. We found that 9-ING-41 potentiates the antitumor effects of gemcitabine (P < 0.05) and cisplatin (P < 0.05) in T24 and HT1376 bladder cancer cells (Fig. 4).

9-ING-41 therapy enhances cytotoxic effect of human immune cells.
To explore whether GSK-3 inhibition potentiates cytotoxic effect of human immune cells in bladder cancer cell lines, we treated T24 and HT1376 bladder cancer cell lines with 9-ING-41 for 48 hours, added the LAK cells at various ratio and measured LDH activity of supernatant to evaluate a cytotoxic effect of immune cells. Our results demonstrate that treatment with 9-ING-41 significantly increased cytotoxic effect of human immune cells in bladder cancer cell lines (Fig. 5).

Discussion
GSK-3β has been identified as a potential therapeutic target in human bladder cancer 11 . Pharmacologic inhibition or genetic depletion of GSK-3β resulted in a decreased viability of bladder cancer cells 11 . In the present study, we tested the GSK-3β inhibitor 9-ING-41, a targeted therapeutic currently being evaluated in a phase 1/2 clinical trial in advanced cancer patients, in bladder cancer cell lines. This is the first report of 9-ING-41 efficacy in bladder cancer. This compound previously demonstrated significant antitumor activity in vitro and in vivo in pre-clinical models of neuroblastoma 14 , lymphoma 15 , glioblastoma 16 , ovarian 12 , pancreatic 17 , renal 10 and breast 18

cancer.
Recently it has been demonstrated that genetic depletion or pharmacologic inhibition of GSK-3β by 9-ING-41 induced cell cycle arrest at G2/M in lymphoma cells 24 . Consistent with the results from the lymphoma study 24 , we found cell cycle blockage at G2/M in 9-ING-41-treated bladder cancer cells, suggesting that GSK-3 inactivation by 9-ING-41 halts progression of mitosis in bladder cancer cells. The exact mechanism by which GSK-3 supports progression of mitosis in cancer cells is unknown. Here, we identified that expression of cyclin B1 and Cdk1, cell cycle regulatory proteins, are affected by GSK-3 inhibition in cancer cells. It has been shown that cyclin B1-Cdk1 complex is a key regulator of mitotic entry 25 . A large number of proteins are phosphorylated by the cyclin B1-Cdk1 complex prior to mitotic entry 25 . We found that expression of cyclin B1 and Cdk1 are downregulated in bladder cancer cell lines treated with 9-ING-41. Our results suggest that GSK-3 positively regulates the expression of cyclin B1 and Cdk1, and treatment with 9-ING-41 leads to cell cycle arrest at G2-phase in bladder cancer cells.
It has been shown that inhibition of GSK-3β leads to apoptosis via p53 activation in p53-wt colon cancer cells 26,27 . Although GSK-3β depletion didn't affect p53-null colon cancer cell lines, it has been demonstrated that GSK-3β silencing sensitized chemoresistant p53-null colon cancer cells to antitumor effects of DNA-damaging drugs 26 . In agreement with the results of colon cancer studies 26 , we found that the GSK-3 inhibitor 9-ING-41 induced apoptosis in p53-wt RT4 bladder cancer cell line, whereas p53-mut HT1376 bladder cancer cells were more resistant to 9-ING-41 therapy which showed mostly growth inhibitory effect. These results suggest that GSK-3 targeted therapy with 9-ING-41 might be an effective treatment for p53-wt bladder cancer. GSK-3β has been shown as a positive regulator of NF-κB-mediated chemoresistance of cancer cells 23 . Here, we demonstrate that GSK-3 inhibitor 9-ING-41 potentiates the antitumor effect of conventional chemotherapeutic drugs in chemoresistant p53-mut T24 and HT1376 bladder cancer cells. Our results identify 9-ING-41 as a candidate for the treatment of p53-wt and p53-mut bladder cancer, and support a rationale to combine 9-ING-41 with gemcitabine or cisplatin, standard of care chemotherapeutic drugs, for metastatic bladder cancer therapy.
Whether autophagy contributes to cancer cell death or represents a mechanism of resistance to anticancer therapy remains unclear 19 . Recent studies demonstrated that GSK-3 inhibits autophagy through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) 28 . It has been shown that inhibition of GSK-3 decreased mTORC1 activity and increased autophagic flux in cancer cells 28 . Moreover, overexpression of either GSK-3α or GSK-3β activates mTORC1 and suppresses autophagy in breast cancer cells 28 . It has been demonstrated that inhibition of GSK-3 induced an autophagic response in pancreatic 29 , prostate 30 and renal cancer 10  www.nature.com/scientificreports www.nature.com/scientificreports/ with previous studies, we found that treatment with 9-ING-41 resulted in autophagy in bladder cancer cells. Importantly, we demonstrated that inhibition of autophagy with chroloquine potentiates the antitumor effect of 9-ING-41 in bladder cancer cells. Our results suggest that potential autophagy-mediated resistance to GSK-3 inhibitor 9-ING-41 could be overcome by using a combination of 9-ING-41 with autophagy inhibitor in human bladder cancer.
Cisplatin-based chemotherapy remains the standard treatment in metastatic bladder cancer patients. Recently, immune checkpoint inhibitors pembrolizumab, atezolizumab, durvalumab, nivolumab and avelumab were www.nature.com/scientificreports www.nature.com/scientificreports/ approved by FDA for the second line setting in metastatic bladder cancer patients who failed cisplatin-based chemotherapy 31 . In the present study, we found that treatment with GSK-3 inhibitor 9-ING-41 enhanced the cytotoxic effect of human immune cells in bladder cancer cell lines. Previously published studies demonstrated that GSK-3 inhibition could lead to upregulation of Fas ligand in cancer cells [32][33][34] which could potentially enhance the sensitivity of cancer cells to cytotoxic effects of human immune cells. Our results demonstrate that 9-ING-41-treated bladder cancer cells are more sensitive to activated human immune cells. Our results suggest that 9-ING-41 therapy might potentiate antitumor immune response in bladder cancer patients.
Overall, the results of the current study provide evidence that treatment with 9-ING-41 could be a potential therapeutic approach to overcome tumor chemo-and immune-resistance in bladder cancer and provide a rationale for the inclusion of patients with advanced bladder cancer in clinical studies of 9-ING-41.

Measurement of cell viability and cell proliferation. Cell viability was measured with a colorimetric
CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer's instructions as described previously 11 . BrdU cell proliferation assay (Calbiochem, Darmstadt, Germany) was carried out according to the manufacturer's instructions 11 . Experiments were performed in three replicates using a flat-bottom 96-well plate (Corning, NY). GI 50 , a concentration of the drug that inhibits the growth of cancer cells by 50%, was calculated using GraphPad Prism 7 (GraphPad, San Diego, CA).

RNA extraction and RT-PCR.
Total cellular RNA was extracted using the SV total RNA Isolation System (Promega, Madison, WI) and first-strand DNA was synthesized using a High Capacity cDNA Reverse Trascription Kit (Applied Biosystems, Waltham, MA) according to the manufacturer's instructions as described previously 11 . Real-time quantitative reverse transcriptase-PCR (RT-PCR) was done in the 7500 Real Time PCR System (Applied Biosystems, Waltham, MA) using pre-designed TaqMan Universal PCR Mastermix (Applied Biosystems, Waltham, MA) targeting human Bcl-2 and XIAP mRNA, and GAPDH was used as endogenous control 11 . Each experiment was repeated three times to confirm reproducibility with the reaction in triplicate wells for each sample. The expression of the target mRNA was quantified relative to that of the GAPDH mRNA 11 . Western immunoblotting analysis. We performed immunoblotting analysis as described previously 11 .
The following antibodies were used: GSK-3β, Cyclin B1, Cdk1, β-actin from Cell Signaling Technology (Danvers, MA); XIAP, PARP from BD Biosciences (Franklin Lakes, NJ). We comply with the digital image and integrity policy.  www.nature.com/scientificreports www.nature.com/scientificreports/ Caspase activation assay. Caspase activation was detected by measuring the activity of caspase-3 (DEVDase) using CaspACE Assay System, Colorimetric (Promega, Madison, WI). According to manufacturer's instruction, labeled p-nitoroaniline (pNA) which was released from the substrate upon cleavage by DEVDase, was measured by a spectrophotometer at 405 nm.
In vitro cytotoxic assay. The study was approved by Niigata University Ethical Committee(IRB No. 2620) and was carried out in accordance to the ethical principals of the Declaration of Helsinki. After obtaining informed consent, blood samples were obtained from healthy human volunteers. Peripheral blood mononuclear cells (PBMCs) were separated using Lymphocyte Separation Solution (Nacalai Tesque, Kyoto, Japan). For lymphokine-activated killer (LAK) induction, PBMCs were suspended at a concentration of 2 × 10 6 cells/ml in RPMI 1640 containing 5% FCS and IL-2 was added at a concentration 2000 U/ml. PBMCs were cultured in 25-cm 2 tissue culture flasks for 4-6 days at 37 °C in a 5% CO 2 atmosphere. T24 and HT1376 cells were treated with 2.5 μM of 9-ING-41 for 48 hours. Untreated cells were used as control. Cytotoxic assay was done using the CytoTox 96 Non-Radioactive Cytotoxic Assay (Promega, Madison, WI) according to the manufacturer's instructions. Released lactate dehydrogenase (LDH) was measured at 490 nM using IMARK microplate reader (Bio-Rad Laboratories, Inc., Tokyo, Japan).

Statistical analysis.
Continuous variables are presented as the mean ± standard deviation (SD). Cell viability assay data were analyzed using one-way ANOVA. Statistical analysis was performed using GraphPad Prism 7.0 software. P < 0.05 was considered statistically significant.

Data availability
All data generated or analyzed during this study are included in this manuscript.